Nanomechanics of Blood Clot and Thrombus Formation

Mechanical properties have been extensively studied in pure elastic or viscous materials; however, most biomaterials possess both physical properties in a viscoelastic component. How the biomechanics of a fibrin clot is related to its composition and the microenvironment where it is formed is not yet fully understood. This review gives an outline of the building mechanisms for blood clot mechanical properties and how they relate to clot function. The formation of a blood clot in health conditions or the formation of a dangerous thrombus go beyond the mere polymerization of fibrinogen into a fibrin network. The complex composition and localization of in vivo fibrin clots demonstrate the interplay between fibrin and/or fibrinogen and blood cells. Studying these protein–cell interactions and clot mechanical properties may represent new methods for the evaluation of cardiovascular diseases (the leading cause of death worldwide), creating new possibilities for clinical diagnosis, prognosis, and therapy. Expected final online publication date for the Annual Review of Biophysics, Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Protein Sequencing, One Molecule at a Time

Despite tremendous gains over the past decade, methods for characterizing proteins have generally lagged behind those for nucleic acids, which are characterized by extremely high sensitivity, dynamic range, and throughput. However, the ability to directly characterize proteins at nucleic acid levels would address critical biological challenges such as more sensitive medical diagnostics, deeper protein quantification, large-scale measurement, and discovery of alternate protein isoforms and modifications and would open new paths to single-cell proteomics. In response to this need, there has been a push to radically improve protein sequencing technologies by taking inspiration from high-throughput nucleic acid sequencing, with a particular focus on developing practical methods for single-molecule protein sequencing (SMPS). SMPS technologies fall generally into three categories: sequencing by degradation (e.g., mass spectrometry or fluorosequencing), sequencing by transit (e.g., nanopores or quantum tunneling), and sequencing by affinity (as in DNA hybridization–based approaches). We describe these diverse approaches, which range from those that are already experimentally well-supported to the merely speculative, in this nascent field striving to reformulate proteomics. Expected final online publication date for the Annual Review of Biophysics, Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Native Mass Spectrometry: Recent Progress and Remaining Challenges

Native mass spectrometry (nMS) has emerged as an important tool in studying the structure and function of macromolecules and their complexes in the gas phase. In this review, we cover recent advances in nMS and related techniques including sample preparation, instrumentation, activation methods, and data analysis software. These advances have enabled nMS-based techniques to address a variety of challenging questions in structural biology. The second half of this review highlights recent applications of these technologies and surveys the classes of complexes that can be studied with nMS. Complementarity of nMS to existing structural biology techniques and current challenges in nMS are also addressed. Expected final online publication date for the Annual Review of Biophysics, Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Lipid–Protein Interactions in Plasma Membrane Organization and Function

Lipid–protein interactions in cells are involved in various biological processes, including metabolism, trafficking, signaling, host–pathogen interactions, and transmembrane transport. At the plasma membrane, lipid–protein interactions play major roles in membrane organization and function. Several membrane proteins have motifs for specific lipid binding, which modulate protein conformation and consequent function. In addition to such specific lipid–protein interactions, protein function can be regulated by the dynamic, collective behavior of lipids in membranes. Emerging analytical, biochemical, and computational technologies allow us to study the influence of specific lipid–protein interactions, as well as the collective behavior of membranes on protein function. In this article, we review the recent literature on lipid–protein interactions with a specific focus on the current state-of-the-art technologies that enable novel insights into these interactions. Expected final online publication date for the Annual Review of Biophysics, Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Chaperonin Mechanisms: Multiple and (Mis)Understood?

The chaperonins are ubiquitous and essential nanomachines that assist in protein folding in an ATP-driven manner. They consist of two back-to-back stacked oligomeric rings with cavities in which protein (un)folding can take place in a shielding environment. This review focuses on GroEL from Escherichia coli and the eukaryotic chaperonin-containing t-complex polypeptide 1, which differ considerably in their reaction mechanisms despite sharing a similar overall architecture. Although chaperonins feature in many current biochemistry textbooks after being studied intensively for more than three decades, key aspects of their reaction mechanisms remain under debate and are discussed in this review. In particular, it is unclear whether a universal reaction mechanism operates for all substrates and whether it is passive, i.e., aggregation is prevented but the folding pathway is unaltered, or active. It is also unclear how chaperonin clients are distinguished from nonclients and what are the precise roles of the cofactors with which chaperonins interact. Expected final online publication date for the Annual Review of Biophysics, Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Variable-Temperature Native Mass Spectrometry for Studies of Protein Folding, Stabilities, Assembly, and Molecular Interactions

The structures and conformational dynamics of proteins, protein complexes, and their noncovalent interactions with other molecules are controlled specifically by the Gibbs free energy (entropy and enthalpy) of the system. For some organisms, temperature is highly regulated, but the majority of biophysical studies are carried out at room, nonphysiological temperature. In this review, we describe variable-temperature electrospray ionization (vT-ESI) mass spectrometry (MS)-based studies with unparalleled sensitivity, dynamic range, and selectivity for studies of both cold- and heat-induced chemical processes. Such studies provide direct determinations of stabilities, reactivities, and thermodynamic measurements for native and non-native structures of proteins and protein complexes and for protein–ligand interactions. Highlighted in this review are vT-ESI-MS studies that reveal 40 different conformers of chymotrypsin inhibitor 2, a classic two-state (native → unfolded) unfolder, and thermochemistry for a model membrane protein system binding lipid and its regulatory protein. Expected final online publication date for the Annual Review of Biophysics, Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Phospholipid Scrambling by G Protein–Coupled Receptors

Rapid flip-flop of phospholipids across the two leaflets of biological membranes is crucial for many aspects of cellular life. The transport proteins that facilitate this process are classified as pump-like flippases and floppases and channel-like scramblases. Unexpectedly, Class A G protein–coupled receptors (GPCRs), a large class of signaling proteins exemplified by the visual receptor rhodopsin and its apoprotein opsin, are constitutively active as scramblases in vitro. In liposomes, opsin scrambles lipids at a unitary rate of >100,000 per second. Atomistic molecular dynamics simulations of opsin in a lipid membrane reveal conformational transitions that expose a polar groove between transmembrane helices 6 and 7. This groove enables transbilayer lipid movement, conceptualized as the swiping of a credit card (lipid) through a card reader (GPCR). Conformational changes that facilitate scrambling are distinct from those associated with GPCR signaling. In this review, we discuss the physiological significance of GPCR scramblase activity and the modes of its regulation in cells. Expected final online publication date for the Annual Review of Biophysics, Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Mapping Enzyme Landscapes by Time-Resolved Crystallography with Synchrotron and X-Ray Free Electron Laser Light

Directly observing enzyme catalysis in real time at the molecular level has been a long-standing goal of structural enzymology. Time-resolved serial crystallography methods at synchrotron and X-ray free electron laser (XFEL) sources have enabled researchers to follow enzyme catalysis and other nonequilibrium events at ambient conditions with unprecedented time resolution. X-ray crystallography provides detailed information about conformational heterogeneity and protein dynamics, which is enhanced when time-resolved approaches are used. This review outlines the ways in which information about the underlying energy landscape of a protein can be extracted from X-ray crystallographic data, with an emphasis on new developments in XFEL and synchrotron time-resolved crystallography. The emerging view of enzyme catalysis afforded by these techniques can be interpreted as enzymes moving on a time-dependent energy landscape. Some consequences of this view are discussed, including the proposal that irreversible enzymes or enzymes that use covalent catalytic mechanisms may commonly exhibit catalysis-activated motions. Expected final online publication date for the Annual Review of Biophysics, Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Chiral Induced Spin Selectivity and Its Implications for Biological Functions

Chirality in life has been preserved throughout evolution. It has been assumed that the main function of chirality is its contribution to structural properties. In the past two decades, however, it has been established that chiral molecules possess unique electronic properties. Electrons that pass through chiral molecules, or even charge displacements within a chiral molecule, do so in a manner that depends on the electron's spin and the molecule's enantiomeric form. This effect, referred to as chiral induced spin selectivity (CISS), has several important implications for the properties of biosystems. Among these implications, CISS facilitates long-range electron transfer, enhances bio-affinities and enantioselectivity, and enables efficient and selective multi-electron redox processes. In this article, we review the CISS effect and some of its manifestations in biological systems. We argue that chirality is preserved so persistently in biology not only because of its structural effect, but also because of its important function in spin polarizing electrons. Expected final online publication date for the Annual Review of Biophysics, Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Enzymology and Dynamics by Cryogenic Electron Microscopy

Cryogenic electron microscopy (cryo-EM) has revolutionized the field of structural biology, particularly in solving the structures of large protein complexes or cellular machineries that play important biological functions. This review focuses on the contribution and future potential of cryo-EM in related emerging applications—enzymatic mechanisms and dynamic processes. Work on these subjects can benefit greatly from the capability of cryo-EM to solve the structures of specific protein complexes in multiple conditions, including variations in the buffer condition, ligands, and temperature, and to capture multiple conformational states, conformational change intermediates, and reaction intermediates. These studies can expand the structural landscape of specific proteins or protein complexes in multiple dimensions and drive new advances in the fields of enzymology and dynamic processes. The advantages and complementarity of cryo-EM relative to X-ray crystallography and nuclear magnetic resonance with regard to these applications are also addressed. Expected final online publication date for the Annual Review of Biophysics, Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.